New Cluster of Plasmid-Located Class 1 Integrons in Vibrio cholerae O1 and a dfrA15 Cassette-Containing Integron in Vibrio parahaemolyticus Isolated in Angola

Daniela Ceccarelli; Anna Maria Salvia; Joana Sami; Piero Cappuccinelli; Mauro Maria Colombo

doi:10.1128/AAC.01310-05

DOI: 10.1128/AAC.01310-05

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FIG. 1.
Map of the 19-kb region on the p3iANG plasmid, clustering together the three integrons and chloramphenicol, kanamycin, and tetracycline resistance genes (approximate sizes; not to scale). The upper and lower lines represent the amplicons obtained by the primer pairs as indicated and oriented by arrows. Primer pairs and resulting amplicons for sequencing the 3.30-kb amplicon are described in the text; the 140-bp junction between the two integrons is indicated by the solid black rectangle. Sequence DQ227350 corresponds to the 3.3-kb 3′CSrev/5′CSrev amplicon. Sequence DQ149925 corresponds to the contiguous 1.5-kb In-F/In-B amplicon.

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TABLE 1.

Vibrio strains under study

Strain	Date of isolation (day/mo/yr)	Place of isolation (Angolan province)	Resistances in addition to common profile^a
V. cholerae O1 582	04/03/92	Luanda	KAN, SUL, TMP, TET
V. cholerae O1 583	09/03/92	Luanda	KAN, SUL, TMP, TET
V. cholerae O1 588	10/03/92	Luanda	ERY, KAN, SUL, TMP, TET
V. cholerae O1 611	26/03/92	Luanda	KAN, SUL, TMP, TET
V. cholerae O1 647	21/04/92	Luanda	KAN, SUL, TMP, TET
V. cholerae O1 817	28/06/94	Bengo (Caxito)	KAN, SUL, TMP, TET
V. cholerae O1 819	28/06/94	Bengo (Dondo)	KAN, SUL, TMP, TET
V. cholerae O1 1383	09/11/94	Benguela	KAN, SUL, TMP, TET
V. cholerae O1 1356	23/11/94	Cuando Cubango	KAN, SUL, TMP, TET
V. cholerae O1 1361	03/05/95	Cabinda	KAN, SUL, TMP, TET
V. cholerae O1 908	16/04/96	Cabinda	ERY, KAN, SUL, TMP, TET
V. parahaemolyticus 570	06/12/91	Luanda	SUL, TMP
V. parahaemolyticus 586	17/03/92	Luanda	SUL, TMP
V. cholerae non-O1 698^b	02/03/93	Bengo River	ERY, SUL, TET
V. cholerae O1 547^b	31/09/94	Bengo River	KAN, SUL, TMP, TET

↵a Abbreviations: AMP, ampicillin; CHL, chloramphenicol; ERY, erythromycin; KAN, kanamycin; PEN, penicillin; STR, streptomycin; SPT, spectinomycin; SUL, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim. The common profile is resistance to AMP, CHL, PEN, STR, and SPT.
↵b Environmental isolate.

TABLE 2.

PCR primers used in this study

Primer and purpose	Sequence (5′ to 3′)	Target gene or description	Amplicon size (bp)	Reference, accession no., and/or chromosomal location^d
Class 1 integron and cassette detection
In-F	GGCATCCAAGCAGCAAG	Class 1 integron	Variable	23
In-B	AAGCAGACTTGACCTGA
Dfr-B^a	TCACCTTCTGGCTCAATGTCG	dfrA15	490	Z83311, bp 374-354
Aad-B^a	GCCGACTACCTTGGTGATCTCG	aadA1	1,476	AY103457, bp 786-765
blaP1-B^a	CTGGTTCATTTCAGATAGCG	blaP1	874	11
Integron cluster mapping and characterization
3′CSrev	AGTCCAGTTCAGACGAA	Integron spacers	Variable	U12338, bp 1433-1416
5′CSrev	GAACGACGAACCTACGG			U12338, bp 4814-4831
Dfr-F^b	GGAGTTATCGGAAATGGCCCAG	Integron spacers	Variable	Z83311, bp 37-58
Bla-F^b	GGCAATCAGCGCTTCCCGTT			AF221899, bp 334-353
Dfr-F2	TTAGCCAAGACTTCGTGT	dfrA15-sul1		AB113114, bp 515-532
Sul1B	GCAAGGCGGAAACCCGCGCC			X12869, bp 1360-1341
Int1A	AAAACCGCCACTGCGCCGTTA	intI1	1,200	12
Int1B	GAAGACGGCTGCACTGAACG
Sul1A^c	GCACGTGCTGTCGAACCTTCA			AY0462763, bp 38844-38865
Aph F	GGCAATCAGGTGCGACAAT	kan	484	AY090559, bp 12978-12997
Aph R	GTGACGACTGAATCCGGTGA			AY090559, bp 13461-13441
CAT1-F	GGCATTTCAGTCAGTTG	cat1	584	V00622, bp 312-328
CAT1-B	CCGCCCTGCCACTCATC			V00622, bp 880-896
TET(G) 1	GCTCGGTGGTATCTCTGCTC	tetG	468	25
TET(G) 2	AGCAACAGAATCGGGAACAC
ICE detection and characterization
Int1-F	GCTGGATAGGTTAAGGGCGG	SXT int	592	8
Int1-B	CTCTATGGGCACTGTCCACATTG
Nint-F	TACCTACAGCAGGAACGGGC	SXT int	258	AF099172, bp 255-274
Nint-B	GCAGCACAGACACCAGACGT			AF099172, bp 513-494
FLOR-F	TTATCTCCCTGTCGTTCCAGCG	floR	526	22
FLOR-2	CCTATGAGCACACGGGGAGC
TMP-F	TGGGTAAGACACTCGTCATGGG	dfr18	389	19
TMP-B	ACTGCCGTTTTCGATAATGTGG
SUL2-F	AGGGGGCAGATGTGATCGC	sul2	625	AY034138, bp 16726-16706
SUL2-B	TGTGCGGATGAAGTCAGCTCC			19
STRA-F	TTGATGTGGTGTCCCGCAATGC	strA	383	19
STRA-B	CCAATCGCAGATAGAAGGCAA
strB-F	GGCACCCATAAGCGTACGCC	strB	470	10
strB-R	TGCCGAGCACGGCGACTACC
dfr1-F	CGAAGAATGGAGTTATCGGG	dfrA1	372	22
dfr1-B	TGCTGGGGATTTCAGGAAAG

↵a Paired with In-F.
↵b Paired by different primer pair combination for mapping the integron cluster (see the text).
↵c To be paired with 5′CSrev.
↵d Primers without references were newly designed.

TABLE 3.

Resistances transferred and class 1 integron cassettes identified in Vibrio strains under study

Strain	Transferred resistances in addition to common profile^a	Class 1 integron cassette(s)
V. cholerae O1 582^d	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b^,^g
V. cholerae O1 583^d	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 611^d	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 1383^d	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 647	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 817	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 819	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 1356	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 1361	KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 588^d	ERY, KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. cholerae O1 908	ERY, KAN, STR, SPT, TET	dfrA15, blaP1, qacH-aadA8^b
V. parahaemolyticus 570		dfrA15^c^,^g
V. parahaemolyticus 586		dfrA15^c^,^g
V. cholerae non-O1 698^d	NC^e
V. cholerae O1 547	NC^f	dfrA15, blaP1, qacH-aadA8^b

↵a Abbreviations: AMP, ampicillin; CHL, chloramphenicol; ERY, erythromycin; KAN, kanamycin; PEN, penicillin; STR, streptomycin; SPT, spectinomycin; SUL, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim. The transferred common profile is resistance to AMP, CHL, SUL, and TMP.
↵b Plasmid located.
↵c Chromosome located.
↵d SXT integrase positive.
↵e NC, not conjugative.
↵f AMP, CHL, and TMP resistance transferred by electroporation.
↵g Confirmed by amplicon sequencing.